1. Field of the Invention
This invention relates to cell encapsulation and, more particularly, to encapsulating living cells in a multi-layer polymeric membrane.
2. Description of Related Art
Microcapsules for biological substances are composed of thin, semi-permeable membranes of cellular dimensions. Microcapsules can be prepared of various polymers and their contents can consist of enzymes, cells and other biological materials. Microcapsules are prepared in such a way as to prevent their contents from leaking out and causing an immunological reaction, but the microcapsules still allow the nutrients and metabolites to exchange freely. This method has found applications primarily in transplantation of foreign materials in vivo without immunosuppression. One example is microencapsulation of hepatocytes for use in bio-assisted liver devices (BLAD). The surface-to-volume ratio of a spherical microcapsule facilitates maximal transport of nutrients, gases, or metabolites exchange across the membrane. In addition, encapsulation of living cells allows better control of the microenvironment for optimal cellular functions via selection of suitable substrate and incorporation of controlled-release features into the local microenvironment. Other physical characteristics such as mass transport, mechanical and chemical stability can also be configured as desired without drastically affecting the functions of the living cells inside the microcapsules.
The commonly used techniques for cell encapsulation are complex coacervation and interfacial precipitation. Complex coacervation involves the electrostatic interaction of two oppositely charged polyelectrolytes. At the right matching charge density, the two poly-ions combine and migrate to form a colloid-rich or water-insoluble phase. The molecular weight and chain conformation parameters of the poly-ions may also play an important role in the complexation process. Interfacial precipitation simply relies on the solidification of a dissolved polymer upon contact with an aqueous phase.
One of the most extensively studied cell encapsulation schemes is one that involves an alginate-gelation complex coacervation method. In this system, alginate, a glycuranan extracted from the brown seaweed algae, can be chelated by calcium or other multivalent counter-ions to form a gel. These early in vivo results with the alginate-polylysine system have not been consistent because of the uncontrolled purity of alginate, and the incorporation of cells into the external membrane. As a result, a 2-step encapsulation was developed to further shield sensitive cells from the extra-capsular environment. The living cells were mixed with sodium alginate and extruded into calcium chloride to form calcium alginate gel droplets. These gel droplets were incorporated into larger alginate gel spheres and then reacted with a poly-amino acid such as poly-L-lysine to form a semi-permeable membrane. Incubating with sodium citrate liquefied the interior to form microcapsules. Unfortunately, the addition of sodium citrate appears to have affected the functions of the cells. Furthermore, the water-soluble alginate and poly-lysine were shown to be not particularly biocompatible as individual polymers, other matrices such as collagen may be better substrates for cellular functions than alginate.
To encapsulate living cells in natural matrices such as collagen, interfacial precipitation has been used. In this method, hydroxylethyhnethacrylate-methylmethacrylate (HEMA-MMA) solution in dimethyl formamide and cell-suspension in collagen or Matrigel were extruded separately through two concentrically configured needles into a precipitating bath containing largely water with a floating layer of dodecane. Polyacrylates are water insoluble that enhances the in vivo stability of the microcapsules. The living cells encapsulated this way (especially with Matrigel) survive well. The interfacial precipitation requires a more elaborate setup than the complex coacervation to control the microcapsule sizes and minimize the contact of cells with organic solvents.
In co-pending U.S. application Ser. No. 09/414,964, filed Oct. 12, 1999, a negatively charged ter-polymer of hydroxyethyl methacrylate-methyl methacrylate-methacrylic acid (HEMA-MMA-MAA) is used to encapsulate cells within a positively charged collagen. The MAA added into the ter-polymer enhances the water solubility of the polymer, allowing the entire encapsulation to be carried out in an aqueous environment. Hence, the complex coacervation method is used while a simple setup provides for easy control of the microcapsule size. The resulting hepatocyte microcapsules exhibit enhanced cellular functions as well as desirable physical characteristics for use in bio-artificial liver. The microcapsules, however, were mechanically unstable as measured by nano-indentation method. After 4 days of static in vitro culture, the microcapsules became weak and breakable upon harsh handling. Attempts at improving the mechanical stability of the microcapsules resulted in tradeoffs with immune-barrier/mass transfer efficiencies and cellular function.
There remains a need for improved microcapsules that exhibit satisfactory mechanical stability in combination with improved immune-barrier/mass transfer efficiencies and cellular function.